Suspected adverse drug reactions of the type 2 antidiabetic drug class dipeptidyl‐peptidase IV inhibitors (DPP4i): Can polypharmacology help explain?

Abstract To interpret the relationship between the polypharmacology of dipeptidyl‐peptidase IV inhibitors (DPP4i) and their suspected adverse drug reaction (ADR) profiles using a national registry. A retrospective investigation into the suspected ADR profile of four licensed DPP4i in the United Kingdom using the National MHRA Yellow Card Scheme and OpenPrescribing databases. Experimental data from the ChEMBL database alongside physiochemical (PC) and pharmacokinetic (PK) profiles were extracted and interpreted. DPP4i show limited polypharmacology alongside low suspected ADR rates. We found a minimal statistical difference between the unique ADR profiles ascribed to the DPP4i except for total ADRs (χ 2; p < .05). Alogliptin consistently showed the highest suspected ADR rate per 1 000 000 items prescribed. Saxagliptin showed the lowest suspected ADR rate across all organ classes but did not reach statistical difference (χ 2; p > .05). We confirmed the Phase III clinical trial data that showed gastrointestinal and skin reactions are the most reported ADRs across the DPP4i class and postulated underlying mechanisms for this based on possible drug interactions. The main pharmacological mechanism behind the ADRs is attributed to interactions with DPP4 activity and/or structure homolog (DASH) proteins which augment the immune‐inflammatory modulation of DPP4.


| INTRODUC TI ON
Type II diabetes is a metabolic disorder primarily associated with insulin sensitivity causing a functional deficit (insulin resistance) which may deteriorate into reduced excretion. Diabetes mellitus is the 9th leading cause of death worldwide 1 and often associated with cardiovascular events such as myocardial infarction or stroke. In the United Kingdom (UK), diabetes is estimated to account for a 10% of the National Health Service (NHS) budget. 2 UK clinical guidance for type II diabetes recommends lifestyle advice and modification as the first-line therapy, followed by metformin as the second-line therapy. 2 Intensification of treatment comprises "add-on" therapies (five are licensed in the UK); Dipeptidyl peptidase-4 inhibitors (DPP4i), pioglitazone, sulfonylureas (SU), sodium-glucose co-transporter 2 inhibitors (SGLT2i) and, glucagon-like peptide-1 (GLP-1) analogs, which can only be initiated under specialist care.
DPP4 is an enzyme that degrades the incretin hormones GLP-1 and glucose-dependent insulinotropic polypeptide (GIP), which serves to control glycaemic levels post-prandially by stimulating insulin synthesis and secretion from pancreatic -cells and reducing glucagon secretion from -cells. [3][4][5] Additionally, GIP/GLP-1 delays gastric emptying and exert central nervous system modulation, thereby increasing satiety and reducing further food intake. DPP4i increases the circulating incretin hormones levels for longer post-prandially, leading to better glycaemic control, and has shown preservation of pancreatic -cell function through increased cell stimulation, proliferation, differentiation, and survival. [6][7][8] Theoretically, this could slow, or reverse disease progression.
DPP4is have preferential effects over other drug classes depending on patient factors. For example, DPP4i's are considered weight neutral, having a negligible risk of hypoglycemia, 4,7 whereas SU and pioglitazone have a higher risk of hypoglycaemic episodes and are associated with weight gain. [9][10][11] Pioglitazone cannot be initiated in patients with heart failure (HF) due to potential complications. 10 SGLT2i's are associated with a higher risk of diabetic ketoacidosis (DKA) and limb and foot amputations. 12 GLP-1 analogs have poor oral bioavailability and are administered via injection.
Adverse drug reactions (ADR) are the unintended side effects of drugs at clinical doses for an indicated disease. ADRs can significantly impact the pharmaceutical management of up to 20% of hospitalized patients' and 25% of outpatient's care. 13 This is associated with a significant cost; associated NHS hospitalizations equate to £380 M per year. 14 Polypharmacology is how a single drug interacts with multiple targets. 15 Side effects can be a result of interactions with both the desired target and/or other targets within the body, potentially causing harm or death. 13 The UK's Medicines and Healthcare products Regulatory Agency Investigation of the polypharmacology of each DPP4i and attributing polypharmacology to ADRs could potentially enable prospective ADR prediction in this drug class or indeed others. 18,19 Furthermore, where an ADR profile is significantly harmful, which may be attributed to its polypharmacology, this may influence clinical guidance or associated clinical decision making. The objective of this research is to ascertain the links (if any) between the polypharmacology of a DPP4i and the reported ADR profile, relative to prescribing levels.

| Physiochemical properties
Physiochemical (PC) properties influence the absorption, distribution, metabolism, and excretion (ADME) of a given drug and are key to determine the activity in vivo. These properties were compared across the DPP4i class to determine whether they influenced the pharmacology and side effect profile of each drug. Physiochemical properties such as pK a (-log 10 K a ; where K a is the acid dissociation constant), hydrogen bond donators/acceptors (HBDs/HBAs), Log 10 P (experimentally measured partition coefficient of a substance between organic and aqueous environments in its neutral form) and cLog 10 P value is associated with more toxicity (<5 is considered toxic). 27 Where parameters were not given in the resources listed, literature searches using SciFinder® for the PK property + drug name were used to gather missing information. [28][29][30]

| Blood-Brain Barrier (BBB) penetration
The threshold of BBB penetration was assigned using; molecular weight < 450 Da; neutral or basic drugs (determined by pK a ); t PSA < 90 Å; <6 hydrogen bond donors; <2 hydrogen bond acceptors; log 10 D 7.4 1-3; and whether it is a P-glycoprotein (P-gp) substrate. The more BBB penetrant properties a drug possesses, the more likely it will cross the BBB. 31

| Pharmacological properties
Pharmacological target searches were completed using drug name searches on ChEMBL (accessed 29/10/2021) 20 ; a database that extracts and standardizes bioactivity data from medicinal chemistry journals and other databases to identify biological interactions. [32][33][34] Median IC 50 values were calculated to negate extremities of values, as IC 50 values were tested across multiple labs at different time points. IC 50 quantitively shows the concentration of a substance required to inhibit a protein by 50%. ChEMBL searches were refined to single protein assays (homosapien) to make the physiological activity as relevant as possible for human use. Those with interactions >100 000 nM were disregarded from reporting due to insignificant physiological relevance.

| Prescribing data
The Openp rescr ibing.net resource provided prescribing data in primary care across England from January 2017 to August 2021. 35 The prescription items (R x ) were collated to standardize the suspected ADRs and to consider differing prescribing rates between drugs within the class. This is different from typical pharmacovigilance studies (that do not consider drug prescribing levels) and enables a surrogate standardization for suspected ADRs to be more readily compared between drug class members.

| Adverse drug reactions (ADR) data
ADR data were collated from the MHRA Yellow Card Scheme Interactive Drug Analysis Profiles (iDAP) 16 with dates set between January 2017 to August 2021 for single active constituents only. The categories reported were system organ classes; fatalities; sub-category terms selected by a threshold ADR rate of 1.5 ADRs/1000000 R x for at least one of the four drugs. The Yellow Card Scheme did not report ADRs for vildagliptin after December 2020 and, therefore, did not fit the scope of this research, which considered ADRs between January 2017 to August 2021.
Vildagliptin was therefore excluded from further analysis due to incomplete datasets.

| Statistical analysis
Chi-squared (χ 2 ) analysis was performed on the standardized ADR/1000000 R x using Microsoft Excel Version 16.55. The test was performed across all four drugs ( Table 2) and then drug vs drug analysis (S1). A p-value of <.05 was set for statistical significance.

| Physiochemical property results
Chemical properties including molecular weight, t PSA and LLE were similar across the class of DPP4i (Table S2). All DPP4i had LLE >5 suggesting they are non-promiscuous inhibitors, with saxagliptin

| Pharmacokinetic results
All reported drugs have once-daily administration at varied doses and have similar half-lives. Notably, linagliptin had a high V d which suggests a higher distribution around tissues in the body, and this may be explained by the Log 10 D 7.4 value being the most lipophilic of the DPP4i studied. Furthermore, linagliptin had the lowest oral bioavailability, significantly higher PPB, and reduced renal clearance.

| Pharmacological properties
Linagliptin had the strongest interaction with DPP4 (1.0 nM), followed by saxagliptin (3.4 nM) and alogliptin (5.3 nM). Sitagliptin showed the weakest activity with DPP4 (18 nM). Linagliptin also showed a stronger affinity for Fibroblast Activation Protein (FAP) (89 nM) as well as activity with M1 receptors (Table 1). Saxagliptin had an inhibitory activity with FAP, but stronger affinity interactions with proteins DPP8 and DPP9. However, these interactions should not be considered in isolation; given that the C max is considerably lower than off-target interactions, they may not be physiologically relevant. Key interactions are shown in Table 1.

| Open prescribing
Prescribing data shows that sitagliptin is the most prescribed DPP4i, with 11.5 million items (R x ), followed by linagliptin (9.4 million R x ), alogliptin (5.6 million R x ), and saxagliptin (1.0 million R x ). Number of prescriptions does not necessarily equate to the number of patients as the drugs are available in a variety of formulated tablet strengths (S4) and multiple tablets may be required to reach the daily indicated dose.

| ADR results
Given the significant difference in prescribed numbers between the four drugs, the ADRs were standardized per 1 000 000 R x for accurate comparison whilst mitigating the risk of misinterpreting unstandardized values.

| DISCUSS ION
The dipeptidyl-peptidase (DPP) family of enzymes includes homologous enzymes DPP2, DPP4, DPP8, DPP9, and FAP. All are N-terminal dipeptide cleaving serine proteases with preferential action where the second amino acid is proline or alanine were the most seen multitarget interactions of the DPP4i's studied (Table 1) Clinical trial data identified gastrointestinal, skin, and infections as common/uncommon ADRs of DPP4i (S5). DPP4i has a wellestablished association with these two ADRs (S1) 41 further confirmed by our study of the Yellow Card Scheme. Our results have confirmed that these are the most reported ADRs and found a minimal statistical difference between the DPP4i drug class.

| Pharmacokinetic and physiochemical properties in relation to pharmacological data and ADRs
All five DPP4i were designed around the two basic amino acids; proline and alanine, giving the four DPP4i some structural similarity, 42 (Figure 1). This in turn generates similar PK and physiochemical properties (Table S2). Notably, linagliptin was the most lipophilic and had the strongest binding interaction with DPP4 and FAP (FAP exhibits the highest sequence identity to DPP4 of the DASH proteins). 37 Regardless of binding affinity with DPP4, there appears to be no correlation with the reported ADR data.

| Gastrointestinal (GI)
One notable ADR across the DPP4i class is acute and chronic pancreatitis with the highest rate associated with linagliptin (2.65 ADRs/1000000 R x ) and lowest rate with alogliptin (1.78). Acute  60 Linagliptin has the highest rate of bullous conditions (4.03) which may be attributed to its moderate affinity with FAP (89 nM); whereas other DPP4i affinity with FAP is significantly weaker. FAP has a defined role in collagen cleavage, 37 which also promotes macrophage adhesion, 61 and therefore the inhibition of FAP may lead to an additional autoantibody-mediated response as well as less macrophage adhesion and activity, which impacts the formation of blisters and skin healing.

| Immunological role (joints and infection)
DPP4, also known as CD26, is involved in amplifying the costimulatory signaling required for T-cell receptor activation and subsequently has an immune modulatory function. 37 Increased DPP4 activity is associated with decreased severity of rheumatic disease due to the dual role of CD26 involving cytokine inhibition and inducing cellular immunity. 36 It is logical therefore that DPP4i are associated with increased arthralgia and joint stiffness, 37 to the extent that an U.S. Food and Drug Administration (FDA) alert (2015) was circulated highlighting the association between DPP4i with severe and disabling joint pain. 62 Alogliptin was reported to have the highest ADR rates associated with musculoskeletal disorders (6.24) and joint

| Cardiovascular
While alogliptin appears to exhibit a difference in cardiac ADR rate over sitagliptin, statistical significance across the DPP4i class was not reached. An FDA safety review has found that type 2 diabetes medicines containing saxagliptin and alogliptin may increase the risk of heart failure, particularly in patients who already have heart or kidney disease. 66 These findings were replicated in previous studies investigating the cardiac safety of alogliptin in type II diabetes, 50,67 which concluded that alogliptin had no additional cardiovascular risk than the placebo. Further trials have conferred that each DPP4i has no additional cardiovascular risk or excessive cardiac toxicity profiles, [47][48][49][50]68 which is particularly important given that patients with type II diabetes are at two-to four-fold increased risk of cardiovascular events. 50 Furthermore, it has also been postulated that this class exerts cardiovascular protection through GLP-1 modulation in vivo, which has well-established cardiovascular effects through coronary artery endothelial cell proliferation and vasculoprotective endothelial progenitor cell stimulation. 69,70 Acknowledging Moreover, patients may stratify side effects in terms of severity and consider some types more concerning than others, leading to higher reporting rates than other ADRs considered less important to report. 17,74 Post-marketing surveillance systems also do not quantify the severity of an ADR; for example, one drug might cause a more severe headache than another, but this is not evaluated by the reporting system and therefore cannot be discussed in this study. This study was able to confirm that the most commonly experienced ADRs in this class were the already-established GI and skin reactions. 41 We have postulated underlying mechanisms for these ADRs in relation to their pharmacological, physiochemical, and pharmacokinetic profiles.
The results demonstrate that the four DPP4i investigated have very similar structural and pharmacokinetic profiles, except for linagliptin, which showed increased lipophilicity and altered pharmacokinetic activity. Regardless of structural differences, the pharmacological activity against DPP4 did not correlate with the total suspected ADRs.
The non-DPP4 targets of the DPP4is of possible physiological relevance were identified as DASH proteins and therefore homologs of the desired protein target, meaning most off-target effects were an augmentation of DPP4-mediated adverse effects. Namely DPP8, DPP9, and FAP, with their role in immune modulation, or the prolonged action of incretin hormones GIP and GLP-1.
Further monitoring of pharmacovigilance schemes and a 5-year review of this data using this methodology may identify the statistical difference between the DPP4is. Continuing monitoring and further research into the cardiac toxicity profile may determine whether there is a protective effect or cardiac damage caused from this therapy.

N O M E N CL ATU R E O F TA RG E T S A N D LI G A N DS
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guide topha rmaco logy.

ACK N OWLED G M ENTS
We thank ChEMBL, MHRA Yellow Card Scheme, and Open Prescribing for providing publicly available data.

FU N D I N G I N FO R M ATI O N
No funding sources to report.

CO N FLI C T O F I NTE R E S T
No known or perceived conflicts of interest are disclosed.

E TH I C S S TATEM ENT
All data collected was publicly available without patient-identifiable information. No ethical approval or consent was required.

DATA AVA I L A B I L I T Y S TAT E M E N T
All underlying data can be found in the supporting materials.